At present, many attempts of gene therapy using a viral vector have been made for the purpose of treatment of cancer or infection as well as treatment of congenital genetic disease. As the viral vector, in many cases, a retroviral vector or an adenoviral vector is conventionally used.
An adeno-associated virus (AAV) is a linear single-stranded DNA virus and can infect cells of a broad range of species including human. AAV also infects non-dividing cells in which differentiation terminates, including blood cells, muscle cells, nerve cells and the like. In addition, since AAV is not pathogenic to human, it has a low risk of adverse effect, and the viral particle of AAV is stable. For these reasons, development of AAV vectors for gene transfer is recently proceeding.
For production of an AAV vector for gene transfer, just like other viral vectors, of elements essential for formation of the viral particle which are present on the wild-type AAV genome the elements that need to be provided in cis and the elements that can be provided in trans are separately introduced into a cell for viral production and expressed in the cell, thereby production of wild-type AAV and self-replication of an AAV vector in a host infected with the AAV vector are prevented (Patent Literature 1).
An established conventional process for producing an AAV vector comprises 1) introduction of an AAV vector plasmid in which an ITR placed at each end of the wild-type AAV genome is left and rep and cap genes are removed, 2) introduction of a plasmid for expression of rep and cap genes to provide Rep and Cap proteins in trans, and since AAV needs provision of supplemental elements from a so-called helper virus such as an adenovirus, a herpesvirus, or a vaccinia virus for formation of the infectious viral particle, 3) infection with an adenovirus (Patent Literature 2).
However, the AAV vector obtained by the above-mentioned process is theoretically contaminated with the adenovirus. The adenovirus contamination needs to be removed or inactivated, which is responsible for decrease in AAV vector titer. In addition, there is a worry that administration of the AAV vector contaminated with adenovirus to a human may cause a side effect such as adenovirus infection or inflammation. Thus, a process for producing an AAV vector comprising, instead of the above-mentioned step 3), step 3′) introduction of a helper plasmid expressing only elements essential for formation of an AAV viral particle among adenovirus-derived elements (Helper-free system) has been developed (Patent Literature 3. The AAV vector produced according to such a process is not contaminated with an adenovirus, and therefore it is excellently safe.
On the other hand, for production of an AAV vector intended for use in the study or clinical field of gene therapy, it is necessary to obtain a viral vector solution with a high titer. For this purpose, a cell line constantly expressing the rep and cap genes was generated based on a human-derived HeLa cell or A549 cell, and attempts to control the expression levels of these genes were made. However, the AAV vector thus obtained does not have enough titer. Therefore, a process for producing an AAV vector with a higher titer is desired.